Abstract
The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H+-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ε domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ε predicted to disrupt inter domain and subunit ε-α crosstalk and therefore coupling. The ε mutant εR105A,R111A,R113A,R115A (ε4A) showed decreased intracellular ATP, slower growth rates and lower molar growth yields on non-fermentable carbon sources. Cellular respiration and metabolism were all accelerated in the mutant strain indicative of dysregulated oxidative phosphorylation. The ε4A mutant exhibited an altered colony morphology and was hypersusceptible to cell wall-acting antimicrobials suggesting defective cell wall biosynthesis. In silico screening identified a novel mycobacterial F-ATP synthase inhibitor disrupting ε’s coupling activity demonstrating the potential to advance this regulation as a new area for mycobacterial F-ATP synthase inhibitor development.
Highlights
The dynamic interaction of the N- and C-terminal domains of mycobacterial F-adenosine triphosphate (ATP) synthase subunit ε is proposed to contribute to efficient coupling of H+-translocation and ATP synthesis
These data showed that the MtεCTD can undergo a switch from a compact, a conformation displayed in the recent structure of the M. smegmatis F1-ATPase[7], to an extended conformation[10], that would allow coupling of c-ring rotation via a spring-like mechanism inside the subunit ε up to the nucleotide-binding and catalytic subunits
In order to determine whether the arginine residues R105, R111, R113 and R115 with the NTD of mycobacterial subunit ε are essential for the crosstalk between both the NTD and the C-terminal domain (CTD), the M. smegmatis mc2155 F-ATP synthase mutant ε4A with the substitution of the four arginine residues inside subunit ε to alanine was engineered
Summary
The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H+-translocation and ATP synthesis. We used a complementary multidisciplinary approach to shed light on the essentiality of crosstalk between the NTD and CTD within mycobacterial subunit ε and the catalytic headpiece for ATP formation by introducing chromosomal missense mutations (R105A, R111A, R113A and R115A) in the C-terminal helix 2 of M. smegmatis mc[2 155] F-ATP synthase subunit ε (ε(R105A,R111A,R113A,R115A) mutant), called ε4A throughout the text, predicted to interrupt inter-domain and subunit ε-α crosstalk and coupling. The cell morphology of the mutant bacilli changed, and the overall cell length was only one-third of the size of wild-type bacteria (WT) Substitution of these amino acids caused a more than 10-fold drop in ATP synthesis and a moderate reduction in ATP hydrolysis of inverted membrane vesicles (IMVs) derived from M. smegmatis ε4A mutant as well as a reduction in the intracellular ATP level of intact bacteria, when grown in minimal media. A new mycobacterial F-ATP synthase inhibitor was discovered
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